(A) Heat map depicting changes in PD-L1 and HLA-I expression from 44 selected compounds out of 484 compounds in the L1100 library and organized by target. PD-L1 and HLA-I levels were assessed by flow cytometry in C8161 cells treated with compounds at the four indicated concentrations and shown as fold change by comparing MFIs of PD-L1 and HLA-I to vehicle-treated cells. Values were excluded and shaded in gray when cell viability was 50% below vehicle-treated controls. Reg and NU are indicated on the left. (B–C) Representative histograms showing expression of PD-L1 (B) and HLA-I (C) in C8161 cells treated with vehicle, 1 μM NU (B), or 1 μM Reg (C). (D–E) Fold change in PD-L1 and HLA-I in C8161 treated with Reg (D) or NU (E) compared to vehicle-treated cells. (F–G) Fold change in PD-L1 (F) or HLA-I (G) in C8161 treated with Reg, NU, or the combination. HTS was performed once and selected candidates were verified. B–G are representative of three independent measurements.

Human melanoma cell lines (n = 27) were treated with Reg (4 μM), NU (2 μM), Vem (1 μM), or a combination of Reg and NU for 48 hours and changes in the expression of PD-L1 (A), CD155 (C), HLA-I (E), CD73 (F), NGFR (H), and CD55 (J) were determined and shown as fold change compared to vehicle-treated cells. Each symbol represents the mean of a technical triplicate. Median and interquartile range are shown. BRAF-mutant cell lines are represented by circles, NRAS-mutant cell lines by diamonds, and BRAF/NRAS wild-type (WT) cell lines by ‘x’, and were separated into groups as indicated. Synergistic (Syn), additive (Add), and antagonistic (Ant) effects of combination Reg and NU treatment were determined for PD-L1 (B), CD155 (D), CD73 (G), NGFR (I), and CD55 (K) in cell lines treated with six concentrations of Reg, NU, and the combination. Cell lines were separated into BRAF- and NRAS-mutant groups as indicated. Synergy was determined using the Chou-Talalay method as described in the materials and methods. Combinatorial effects in some cell lines were unclear and were not determined (N/D). All results are representative of at least two independent measurements. Statistical analyses accompanying A, C, E, F, H, and J are shown in .